Cloning and expression of Bacillus thuringiensis toxin gene encoding a protein toxic to beetles of the order Coleoptera

ABSTRACT

The toxin gene encoding a protein toxic to beetles of the order Coleoptera, named M-7, has been cloned and expressed. M-7 is a novel Bacillus thuringiensis strain which has been deposited with a recognized culture repository. The microbe is now known as B. thuringiensis strain san diego.

CROSS REFERENCE TO RELATED APPLICATION

This is a continuation-in-part of our copending application Ser. No.767,227, filed on Aug. 16, 1985.

BACKGROUND OF THE INVENTION

The spore-forming microorganism Bacillus thuringiensis (Bt) produces thebest-known insect toxin. The toxin is a protein, designated asδ-endotoxin. It is synthesized by the Bt sporulating cell. The toxin,upon being ingested in its crystalline form by susceptible insectlarvae, is transformed into biologically active moieties by the insectgut juice proteases. The primary target is insect cells of the gutepithelium, which are rapidly destroyed. Experience has shown that theactivity of the Bt toxin is so high that only nanogram amounts arerequired to kill susceptible insect larvae.

The reported activity spectrum of Bt covers insect species within theorder Lepidoptera, which is a major insect problem in agriculture andforestry. The activity spectrum also includes the insect order Diptera,wherein reside some species of mosquitoes and blackflies. See Couch, T.L., (1980) "Mosquito Pathogenicity of Bacillus thuringiensis var.israelensis," Developments in Industrial Microbiology 22:61-67; Beegle,C. C., (1978) "Use of Entomogenous Bacteria in Agroecosystems,"Developments in Industrial Microbiology, 20:97-104.

Kreig et al., Z. ang. Ent. (1983) 96:500-508, describe a Bt isolatenamed Bacillus thuringiensis var. tenebrionis, which is reportedlyactive against two beetles of the order Coleoptera. These are Coloradopotato beetle, Leptinotarsa decemlineata, and Agelastica alni. This isthe only known Bt isolate reported to contain such activity; allpreviously identified Bt strains have had activity against caterpillars(order Lepidoptera) or larvae of certain flies (order Diptera).

The Krieg et al. Bt isolate is not available for side-by-side comparisonwith the Bt isolate used as the source of the novel Bt gene of thesubject invention. Therefore, since the Krieg et al. Bt isolate is notavailable to the public, the Krieg et al. publication is not a validpatent law reference under U.S. law.

BRIEF SUMMARY OF THE INVENTION

Disclosed and claimed is the cloning and expression of the toxin genetoxic to beetles of the order Coleoptera. The toxin produced by thecloned gene has activity against beetles of the order Coleoptera but notagainst Trichoplusia ni, Spodoptera exigua or Aedes aegypti. Included inthe Coleoptera are various Diabrotica species (family Chrysomelidae)that are responsible for large agricultural losses. For example, D.undecimpunctata (western spotted cucumber beetle), D. longicornis(northern corn rootworm), D. virgitera (western corn rootworm), and D.undecimpunctata howardi (southern corn rootworm).

DETAILED DESCRIPTION OF THE INVENTION

The Bacillus thuringiensis isolate used as the source of the toxin geneof the subject invention, designated "M-7," is unusual in having aunique parasporal body (crystal) which under phase contrast microscopyis dark in appearance with a flat, square configuration.

A subculture of B. thuringiensis M-7, now known as B. thuringiensisstrain san diego (B.t.sd) has been deposited in the permanent collectionof the Northern Regional Research Laboratory, U.S. Department ofAgriculture, Peoria, Ill., USA on Feb. 27, 1985. The culture wasassigned the accession number NRRL B-15939 by the repository. Thisdeposit is available to the public upon the grant of a patent disclosingit. The deposit is also available as required by foreign patent laws incountries wherein counterparts of the subject application, or itsprogeny, are filed. However, it should be understood that theavailability of a deposit does not constitute a license to practice thesubject invention in derogation of patent rights granted by governmentalaction.

B. thuringiensis strain san diego, NRRL B-15939, can be cultured usingstandard art media and fermentation techniques. Upon completion of thefermentation cycle, the bacteria can be harvested by first separatingthe Bt spores and crystals from the fermentation broth by means wellknown in the art. The DNA (chromosomal and plasmid) from the cells canbe isolated by standard procedures and purified by procedures well knownin the art. For example, such standard procedures are disclosed inManiatis et al., Molecular Cloning (1982), Cold Spring HarborLaboratory.

The purified DNA then can be digested with a suitable restrictionendonuclease.

A gene bank of B.t.sd DNA then can be constructed. In the subjectinvention, the purified B.t.sd DNA, obtained as described above, wasdigested with the restriction endonuclease BamHI and cloned into theBamHI site of the well-known and available plasmid pBR322.

Once the gene bank of B.t.sd DNA was constructed, it then becamenecessary to construct a DNA probe to screen the gene bank. Theconstruction of this critical DNA probe was initiated by the isolationof M-7 toxin crystals fron a culture of B.t.sd.

The recovered M-7 toxin crystals were purified by standard proceduresand then digested with trypsin to produce peptide fragments. The aminoacid sequences of several of these tryptic fragments was determined bystandard procedures. Subsequently, after selection of certain sequences,a probe was chemically synthesized by known means. The resulting probewas labelled and hybridized by procedures known in the art. The netresult was the detection of positive clones, i.e., those that hybridizedto the constructed probe.

A representative of the positive clones was subjected to a western blotusing rabbit anti M-7 crystal antiserum developed by standardprocedures. Confirmation of the success of the cloning and expression ofM-7 toxin was obtained when a positive reaction was observed with thepositive clone and the antibody against M-7 toxin crystal.

The recombinant plasmids isolated from representative positive cloneswere found to have a 5.8 kb DNA fragment inserted into the BamHI site.This 5.8 kb DNA fragment was excised from a representative positiveclone (pCH-B3) with BamHI, purified, and then subcloned into the BamHIsite of the known and available plasmid pR01614 (J. Bact. [1982] 150:60;U.S. Pat. No. 4,374,200). Plasmid pR01614 is available from the NorthernRegional Research Laboratory, address below, where its deposit number isNRRL B-12127. The plasmid is derived from pBR322 and has unique HindIII,BamHI, and SalI and PvuII restriction sites; a PstI insertion includesthe carbenicillin resistance gene and a P. aeruginosa replicationsystem. Pseudomonas fluorescens was transformed with this constructedshuttle vector and the expression of M-7 toxin was verified by itsidentification on a western blot.

Plasmid pCH-B3, or plasmid pR01614 with the 5.8 kb fragment insert, canbe recovered from their bacterial hosts by well-known procedures, e.g.,using the cleared lysate-isopycnic density gradient procedures. Ifdesired, the 5.8 kb fragment can be excised from pR01614 by digestionwith BamHI and cloned into a different vector for transformation intoanother host. These procedures are all well known to persons skilled inthe art.

Plasmid pCH-B3, in an E. coli host, was deposited with the ARS PatentCollection, Culture Collection Research-Fermentation Laboratory,Northern Regional Research Center, Peoria, Ill. 61604. The deposit wasmade in the permanent collection of the repository to be maintained bythe repository for at least 30 years. The deposit was made on July 18,1985, and given the accession number NRRL B-15981. A subculture isavailable to the public upon the grant of a patent disclosing thedeposit. The deposit is also available as required by foreign patentlaws in countries wherein counterparts of the subject application, orits progeny, are filed. However, it should be understood that theavailability of a deposit does not constitute a license to practice thesubject invention in derogation of patent rights granted by governmentalaction.

The toxin gene of the subject invention can be introduced into a widevariety of microbial hosts. Expression of the toxin gene (M-7) results,directly or indirectly, in the intracellular production and maintenanceof the pesticide. With suitable hosts, e.g., Pseudomonas, the microbescan be applied to the situs of beetles of the order Coleoptera wherethey will proliferate and be ingested by the susceptible beetles. Theresult is a control of the unwanted beetles. Alternatively, the microbehosting the toxin M-7 gene can be treated under conditions that prolongthe activity of the toxin produced in the cell. The treated cell thencan be applied to the environment of target pest(s). The resultingproduct retains the toxicity of the M-7 toxin.

Where the M-7 toxin gene is introduced via a suitable vector into amicrobial host, and said host is applied to the environment in a livingstate, it is essential that certain host microbes be used. Microorganismhosts are selected which are known to occupy the "phytosphere"(phylloplane, phyllosphere, rhizosphere, and/or rhizoplane) of one ormore crops of interest. These microorganisms are selected so as to becapable of successfully competing in the particular environment (cropand other insect habitats) with the wild-type microorganisms, providefor stable maintenance and expression of the gene expressing thepolypeptide pesticide, and, desirably, provide for improved protectionof the pesticide from environmental degradation and inactivation.

A large number of microorganisms are known to inhabit the phylloplane(the surface of plant leaves) and/or the rhizosphere (the soilsurrounding plant roots) of a wide variety of important crops. Thesemicroorganisms include bacteria, algae, and fungi. Of particularinterest are microorganisms, such as bacteria, e.g., genera Pseudomonas,Erwinia, Serratia, Xanthomonas, Streptomyces, Rhizobium,Rhodopseudomonas, Agrobacterium, Acetobacter, Lactobacillus,Arthrobacter, Azotobacter, Leuconostoc, and Alcaligenes; fungi,particularly yeast, e.g., genera, Saccharomyces, Cryptococcus,Kluyveromyces, Sporobolomyces, Rhodotorula, and Aureobasidium. Ofparticular interest are such phytosphere bacterial species asPseudomonas syringae, Pseudomonas fluorescens, Serratia marcescens,Acetobacter xylinum, Agrobacterium tumefaciens, Rhodopseudomonasspheroides, Xanthomonas campestris, Rhizobium melioti, Alcaligenesentrophus, and Azotobacter vinlandii; and phytosphere yeast species suchas Rhodotorula rubra, R. glutinis, R. marina, R. aurantiaca,Cryptococcus albidus, C. diffluens, C. laurentii, Saccharomyces rosei,S. pretoriensis, S. cerevisiae, Sporobolomyces roseus, S. odorus,Kluyveromyces veronae, and Aureobasidium pollulans. Of particularinterest are the pigmented microorganisms.

A wide variety of ways are available for introducing the M-7 geneexpressing the toxin into the microorganism host under conditions whichallow for stable maintenance and expression of the gene. One can providefor DNA constructs which include the transcriptional and translationalregulatory signals for expression of the toxin gene, the toxin geneunder their regulatory control and a DNA sequence homologous with asequence in the host organism, whereby integration will occur, and/or areplication system which is functional in the host, whereby integrationor stable maintenance will occur.

The transcriptional initiation signals will include a promoter and atranscriptional initiation start site. In some instances, it may bedesirable to provide for regulative expression of the toxin, whereexpression of the toxin will occur only after release into theenvironment. This can be achieved with operators or a region binding toan activator or enhancers, which are capable of induction upon a changein the physical or chemical environment of the microorganisms. Forexample, a temperature sensitive regulatory region may be employed,where the organisms may be grown up in the laboratory without expressionof a toxin, but upon release into the environment, expression wouldbegin. Other techniques may employ a specific nutrient medium in thelaboratory, which inhibits the expression of the toxin, where thenutrient medium in the environment would allow for expression of thetoxin. For translational initiation, a ribosomal binding site and aninitiation codon will be present.

Various manipulations may be employed for enhancing the expression ofthe messenger, particularly by using an active promoter, as well as byemploying sequences, which enhance the stability of the messenger RNA.The initiation and translational termination region will involve stopcodon(s), a terminator region, and optionally, a polyadenylation signal.

In the direction of transcription, namely in the 5' to 3' direction ofthe coding or sense sequence, the construct will involve thetranscriptional regulatory region, if any, and the promoter, where theregulatory region may be either 5' or 3' of the promoter, the ribosomalbinding site, the initiation codon, the structural gene having an openreading frame in phase with the initiation codon, the stop condon(s),the polyadenylation signal sequence, if any, and the terminator region.This sequence as a double strand may be used by itself fortransformation of a microorganism host, but will usually be includedwith a DNA sequence involving a marker, where the second DNA sequencemay be joined to the toxin expression construct or may be combined as aseparate DNA fragment with the toxin expression construct duringintroduction of the DNA into the host.

By a marker is intended a structural gene which provides for selectionof those hosts which have been modified or transformed. The marker willnormally provide for selective advantage, for example, providing forbiocide resistance, e.g., resistance to antibiotics or heavy metals;complementation, so as to provide prototrophy to an auxotrophic host, orthe like. Preferably, complementation is employed, so that the modifiedhost may not only be selected, but may also be competitive in the field.One or more markers may be employed in the development of theconstructs, as well as for modifying the host. The organisms may befurther modified by providing for a competitive advantage against otherwild-type microorganisms in the field. For example, genes expressingmetal chelating agents, e.g., siderophores, may be introduced into thehost along with the structural gene expressing the toxin. In thismanner, the enhanced expression of a siderophore may provide for acompetitive advantage for the toxin producing host, so that it mayeffectively compete with the wild-type microorganisms and stably occupya niche in the environment of the vegetation to be protected.

Where no functional replication system is present, the construct willalso include a sequence of at least 50 bp, preferably at least about 100bp, and usually not more than about 1000 bp of a sequence homologouswith a sequence in the host. In this way, the probability of legitimaterecombination is enhanced, so that the gene will be integrated into thehost and stably maintained by the host. Desirably, the toxin gene willbe in close proximity to the gene prqviding for complementation as wellas the gene providing for the competitive advantage. Therefore, in theevent that the toxin gene is lost, the resulting organism will be likelyto also lose the complementing gene and/or the gene providing for thecompetitive advantage, so that it will be unable to compete in theenvironment with the gene retaining the intact construct.

A large number of transcriptional regulatory regions are available froma wide variety of microorganism hosts, such as bacteria, bacteriophage,cyanobacteria, algae, fungi, and the like. Various transcriptionalregulatory regions include the regions associated with the trp gene, lacgene, gal gene, the lambda left and right promoters, the Tac promoter,the naturally-occurring promoters associated with the toxin gene, wherefunctional in the host. See for example, U.S. Pat. Nos. 4,332,898;4,342,832 and 4,356,270. The termination region may be the terminationregion normally associated with the transcriptional initiation region ora different transcriptional initiation region, so long as the tworegions are compatible and functional in the host.

Where stable episomal maintenance or integration is desired, a plasmidwill be employed which has a replication system which is functional inthe host. The replication system may be derived from the chromosome, anepisomal element normally present in the host or a different host, or areplication system from a virus which is stable in the host. A largenumber of plasmids are available, such as pBR322, pACYC184, RSF1010,pR01614, and the like. See for example, Olson et al., (1982) J.Bacteriol. 150:6069, and Bagdasarian et al., (1981) Gene 16:237, andU.S. Pat. Nos. 4,356,270; 4,362,817; and 4,371,625.

The M-7 gene can be introduced between the transcriptional andtranslational initiation region and the transcriptional andtranslational termination region, so as to be under the regulatorycontrol of the initiation region. This construct will be included in aplasmid, which will include at least one replication system, but mayinclude more than one, where one replication system is employed forcloning during the development of the plasmid and the second replicationsystem is necessary for functioning in the ultimate host. In addition,one or more markers may be present, which have been describedpreviously. Where integration is desired, the plasmid will desirablyinclude a sequence homologous with the host genome.

The transformants can be isolated in accordance with conventional ways,usually employing a selection technique, which allows for selection ofthe desired organism as against ummodified organisms or transferringorganisms, when present. The trausformants then can be tested forpesticidal activity.

Preferred hosts, particularly those in the phytosphere, will havecertain characteristics which enhance the environmental stability of thetoxins in the host. Protective qualities include a low level ofproteolytic degradation, thick cell walls, pigmentation, and the like.Other characteristics of interest for the host include leaf affinity,lack of mammalian toxicity, attractiveness to pests for ingestion, easeof handling and storage, rate of proliferation in the field,competitiveness, and the like.

In the field applications, the transformant strain will be applied toits natural habitat, such as the rhizosphere or phylloplane of the plantto be protected from the pest. The transformant strain will grow in itsnatural habitat, while producing the M-7 toxin which will be absorbedand/or ingested by the larvae or adult pest, or have a toxic effect onthe ova. The persistence of the microorganisms will provide forlong-term protection of the vegetation, although repetitiveadministrations may be required from time to time. The organism may beapplied by spraying, soaking, injection into the soil, seed coating,seedling coating or spraying, or the like. Where administered in thefield, generally concentrations of the organism will be from 10⁶ to 10¹⁰cells/ml, and the volume applied per hectare will be generally fromabout 0.1 oz to 2 lbs or more. Where administered to a plant part, theconcentration of the organism will usually be from 10³ to 10⁶ cells/cm².

Suitable host cells, where the pesticide-containing cells will betreated to prolong the activity of the toxin in the cell when the thendead cell is applied to the environment of target pest(s), may includeeither prokaryotes or eukaryotes, normally being limited to those cellswhich do not produce substances toxic to higher organisms, such asmammals. However, organisms which produce substances toxic to higherorganisms could be used, where the toxin is unstable or the level ofapplication sufficiently low as to avoid any possibility of toxicity toa mammalian host. As hosts, of particular interest will be theprokaryotes and lower eukaryotes, such as fungi. Illustrativeprokaryotes, both Gramnegative and -positive, includeEnterobacteriaceae, such as Escherichia, Erwinia, Shigella, Salmonella,and Proteus; Bacillaceae; Rhizobiaceae, such as Rhizobium; Spirillaceae,such as photobacterium, Zymomonas, Serratia, Aeromonas, Vibrio,Desulfovibrio, Spirillum; Lactobacillaceae; Pseudomonadaceae, such asPseudomonas and Acetobacter; Azotobacteraceae and Nitrobacteraceae.Among eukaryotes are fungi, such as Phycomycetes and Ascomycetes, whichincludes yeast, such as Saccharomyces and Schizosaccharomyces; andBasidiomycetes yeast, such as Rhodotorula, Aureobasidium,Sporobolomyces, and the like.

Characteristics of particular interest in selecting a host cell forpurposes of production include ease of introducing the M-7 gene into thehost, availability of expression systems, efficiency of expression,stability of the pesticide in the host, and the presence of auxiliarygenetic capabilities. Characteristics of interest for use as a pesticidemicrocapsule include protective qualities for the pesticide, such asthick cell walls, pigmentation, and intracellular packaging or formationof inclusion bodies; leaf affinity; lack of mammalian toxicity;attractiveness to pests for ingestion; ease of killing and fixingwithout damage to the toxin; and the like. Other considerations includeease of formulation and handling, economics, storage stability, and thelike.

Host organisms of particular interest include yeast, such as Rhodotorulasp., Aureobasidium sp., Saccharomyces sp., and Sporobolomyces sp.;phylloplane organisms such as Pseudomonas sp., Erwinia sp. andFlavobacterium sp.; or such other organisms as Escherichia,Lactobacillus sp., Bacillus sp., and the like. Specific organismsinclude Pseudomonas aeruginosa, Pseudomonas fluorescens, Saccharomycescerevisiae, Bacillus thuringiensis, Escherichia coli, Bacillus subtilis,and the like.

The cell will usually be intact and be substantially in theproliferative form when killed, rather than in a spore form, although insome instances spores may be employed.

The cells may be inhibited from proliferation in a variety of ways, solong as the technique does not deleteriously affect the properties ofthe pesticide, nor diminish the cellular capability in protecting thepesticide. The techniques may involve physical treatment, chemicaltreatment, changing the physical character of the cell or leaving thephysical character of the cell substantially intact, or the like.

Various techniques for inactivating the host cells include heat, usually50° C. to 70° C.; freezing; UV irradiation; lyophilization; toxins,e.g., antibiotics; phenols; anilides, e.g., carbanilide andsalicylanilide; hydroxyurea; quaternaries; alcohols; antibacterial dyes;EDTA and amidines; non-specific organic and inorganic chemicals, such ashalogenating agents, e.g., chlorinating, brominating or iodinatingagents; aldehydes, e.g., glutaraldehyde or formaldehyde; toxic gases,such as ozone and ethylene oxide; peroxide; psoralens; desiccatingagents or the like, which may be used individually or in combination.The choice of agent will depend upon the particular pesticide, thenature of the host cell, the nature of the modification of the cellularstructure, such as fixing and preserving the cell wall with crosslinkingagents, or the like.

The cells generally will have enhanced structural stability which willenhance resistance to environmental degradation in the field. Where thepesticide is in a proform, the method of inactivation should be selectedso as not to inhibit processing of the proform to the mature form of thepesticide by the target pest pathogen. For example, formaldehyde willcrosslink proteins and could inhibit processing of the proform of apolypeptide pesticide. The method of inactivation or killing retains atleast a substantial portion of the bioavailability or bioactivity of thetoxin.

The cellular host containing the M-7 pesticidal gene may be grown in anyconvenient nutrient medium, where the DNA construct provides a selectiveadvantage, providing for a selective medium so that substantially all orall of the cells retain the M-7 gene. These cells may then be harvestedin accordance with conventional ways. Alternatively, the cells can befixed prior to harvesting.

The method of treating the host organism containing the toxin canfulfill a number of functions. First, it may enhance structuralintegrity. Second, it may provide for enhanced proteolytic stability ofthe toxin, by modifying the toxin so as to reduce its suceptibility toproteolytic degradation and/or by reducing the proteolytic activity ofproteases naturally present in the cell. The cells are preferablymodified at an intact stage and when there has been a substantialbuildup of the toxin protein. These modifications can be achieved in avariety of ways, such as by using chemical reagents having a broadspectrum of chemical reactivity. The intact cells can be combined with aliquid reagent medium containing the chemical reagents, with or withoutagitation, at temperatures in the range of about -10 to 60° C. Thereaction time may be determined empirically and will vary widely withthe reagents and reaction conditions. Cell concentrations will vary fromabout 10² to 10¹⁰ per ml.

Of particular interest as chemical reagents are halogenating agents,particularly halogens of atomic no. 17-80. More particularly, iodine canbe used under mild conditions and for sufficient time to achieve thedesired results. Other suitable techniques include treatment withaldehydes, such as formaldehyde and glutaraldehyde; anti-infectives,such as zephiran chloride and cetylpyridinium chloride; alcohols, suchas isopropyl and ethanol; various histologic fixatives, such as Bouin'sfixative and Helly's fixative (see Humason, Gretchen L., Animal TissueTechniques, W. H. Freeman and Company, 1967); or a combination ofphysical (heat) and chemical agents that prolong the activity of thetoxin produced in the cell when the cell is applied to the environmentof the target pest(s).

For halogenation with iodine, temperatures will generally range fromabout 0° to 50° C., but the reaction can be conveniently carried out atroom temperature. Conveniently, the iodination may be performed usingtriiodide or iodine at 0.5 to 5% in an acidic aqueous medium,particularly an aqueous carboxylic acid solution that may vary fromabout 0.5-5 M. Conveniently, acetic acid may be used, although othercarboxylic acids, generally of from about 1 to 4 carbon atoms, may alsobe employed. The time for the reaction will generally range from lessthan a minute to about 24 hr, usually from about 1 to 6 hr. Any residualiodine may be removed by reaction with a reducing agent, such asdithionite, sodium thiosulfate, or other reducing agent compatible withultimate usage in the field. In addition, the modified cells may besubjected to further treatment, such as washing to remove all of thereaction medium, isolation in dry form, and formulation with typicalstickers, spreaders, and adjuvants generally utilized in agriculturalapplications, as is well known to those skilled in the art.

Of particular interest are reagents capable of crosslinking the cellwall. A number of reagents are known in the art for this purpose. Thetreatment should result in enhanced stability of the pesticide. That is,there should be enhanced persistence or residual activity of thepesticide under field conditions. Thus, under conditions where thepesticidal activity of untreated cells diminishes, the activity oftreated cells remains for periods of from 1 to 3 times longer.

The cells may be formulated in a variety of ways. They may be employedas wettable powders, granules or dusts, by mixing with various inertmaterials, such as inorganic minerals (phyllosilicates, carbonates,sulfates, phosphates, and the like) or botanical materials (powderedcorncobs, rice hulls, walnut shells, and the like). The formulations mayinclude spreader-sticker adjuvants, stabilizing agents, other pesticidaladditives, or surfactants. Liquid formulations may be aqueous-based ornon-aqueous and employed as foams, gels, suspensions, emulsifiableconcentrates, or the like. The ingredients may include rheologicalagents, surfactants, emulsifiers, dispersants, or polymers.

The pesticidal concentration will vary widely depending upon the natureof the particular formulation, particularly whether it is a concentrateor to be used directly. The pesticide will be present in at least 1% byweight and may be 100% by weight. The dry formulations will have fromabout 1-95% by weight of the pesticide while the liquid formulationswill generally be from about 1-60% by weight of the solids in the liquidphase. The formulations will generally have from about 10² to about 10⁴cells/mg. These formulations will be administered at about 50 mg (liquidor dry) to 1 kg or more per hectare.

The formulations can be applied to the environment of the pest(s), e.g.,plants, soil or water, by spraying, dusting, sprinkling or the like.

Following are examples which illustrate procedures, including the bestmode, for practicing the invention. These examples should not beconstrued as limiting. All percentages are by weight and all solventmixture proportions are by volume unless otherwise noted.

EXAMPLE 1 Culturing B. thuringiensis strain san diego NRRL B-15939.

A subculture or starter culture of B. thuringiensis strain san diegoNRRL B-15939 can be used to inoculate the following medium, known as LBbroth:

    ______________________________________                                        Tryptone       10            gm                                               Yeast extract  5             gm                                               NaCl           5             gm                                               5 N NaOH       0.6           ml                                               Water          1000          ml                                               ______________________________________                                    

As per standard microbiological techniques, the above medium would besterilized prior to inoculation and the inoculations would be done usingaseptic procedures. The M-7 cells are grown for 3-4 days at 30° C.

A detailed procedure is as follows:

A series of 150 ml Erlenmeyer flasks containing sterile PWYE medium(peptone 5.0%; yeast extract 0.1%; NaCl 0.5% in 1 liter of water; adjustpH to 7.5) are inoculated from a petri plate culture of B. thuringiensisstrain san diego, NRRL B-15939. The flasks are incubated at 30° C. on arotary shaker (200 rpm) overnight. From this starter culture, 300 ml ofLB broth in a 2 liter flask is inoculated using 7.5 ml of the starter.The LB-broth flasks are incubated under the same conditions as thestarter, but are harvested after 4 days.

The above procedure can be readily scaled up to large fermentors byprocedures well known in the art.

The Bt spores and crystals, obtained in the above fermentation, can beisolated by procedures well known in the art. A frequently-usedprocedure is to subject the harvested fermentation broth to separationtechniques, e.g., centrifugation.

EXAMPLE 2 Cloning and Expression of M-7 Toxin Gene

Total DNA (chromosomal and plasmid) was isolated from the M-7 cells ofExample 1 and purified by standard procedures. The resulting purifiedDNA was digested with the restriction endonuclease BamHI, using thesupplier's instruction. The digested DNA was then cloned into the BamHIsite of the well-known plasmid pBR322 to give a gene bank of M-7 DNA.This cloning procedure was done following standard well-knownprocedures.

A DNA probe to screen the gene bank was obtained as follows: M-7crystals were isolated from a culture grown in NYSM medium (10 gmtryptone, 5 gm NaCl, 5 gm yeast extract, 2 gm MgSO₄.7H₂ O, 1000 mlwater, pH 7.5) overnight at 30° C. The purified crystals were dissolvedin 8 M urea, 0.1 M glycine, pH 8.2 and digested with trypsin overnightat room temperature. The resulting peptide fragments were separated on aC₄ reverse phase high pore column with a 180 min gradient of 91%solution A (0.1% trifluoroacetic acid in H₂ O) to 40% solution A in 0.1%trifluoroacetic acid in acetonitrile. The aminoacid sequences of severaltryptic fragments were obtained and a sequence of 6 aminoacids wereselected for synthesis of a mixed probe, 17 bases in length, with aredundancy of 32.

The probe was end-labeled with polynucleotide kinase and [γ-³² P]ATP andhybridized to bacterial colonies containing recombinant plasmids asconstructed for the M-7 gene bank. The colony filters were preparedaccording to Hanahan and Meselson (1980) Gene 10:63-67. Positivecolonies were identified by autoradiography. The recombinant plasmidsisolated from seven positive clones (pCH-B3 as representative) werefound to have a 5.8 kb (kilobase pairs) DNA fragment inserted into theBamHI site.

A western blot (Burnette, W. N. [1981] Anal. Biochem. 112:195) of pCH-B3was performed on an SDS-PAGE of an overnight culture, using rabbitanti-M-7 crystal anti-serum. A protein of about 86 kilodalton wasidentified. The clone pCH-B3, therefore, contains an M-7 DNA fragmentthat encodes for a protein having serological identity with the proteinfrom the M-7 crystals. The recombinant protein may be bigger than thetoxin from solubilized M-7 crystals because of unavailability oftranscriptional and/or translational stop signals in the given plasmidconstruction.

The nucleotide sequence encoding the B.t.sd toxin gene is shown in TableA. The deduced amino acid sequence is shown in Table B.

As is well known in the art, the amino acid sequence of a protein isdetermined by the nucleotide sequence of the DNA. Because of theredundancy of the genetic code, i.e., more than one coding nucleotidetriplet (codon) can be used for most of the amino acids used to makeproteins, different nucleotide sequences can code for a particular aminoacid. Thus, the genetic code can be depicted as follows:

    ______________________________________                                        Phenylalanine (Phe)                                                                        TTK      Histidine (His)                                                                              CAK                                      Leucine (Leu)                                                                              XTY      Glutamine (Gln)                                                                              CAJ                                      Isoleucine (Ile)                                                                           ATM      Asparagine (Asn)                                                                             AAK                                      Methionine (Met)                                                                           ATG      Lysine (Lys)   AAJ                                      Valine (Val) GTL      Aspartic acid (Asp)                                                                          GAK                                      Serine (Ser) QRS      Glutamic acid (Glu)                                                                          GAJ                                      Proline (Pro)                                                                              CCL      Cysteine (Cys) TGK                                      Threonine (Thr)                                                                            ACL      Tryptophan (Trp)                                                                             TGG                                      Alanine (Ala)                                                                              GCL      Arginine (Arg) WGZ                                      Tyrosine (Tyr)                                                                             TAK      Glycine (Gly)  GGL                                      Termination signal                                                                         TAJ                                                              ______________________________________                                    

Key: Each 3-letter deoxynucleotide triplet corresponds to atrinucleotide of mRNA, having a 5'-end on the left and a 3'-end on theright. All DNA sequences given herein are those of the strand whosesequence corresponds to the mRNA sequence, with thymine substituted foruracil. The letters stand for the purine or pyrimidine bases forming thedeoxynucleotide sequence.

A=adenine

G=guanine

C=cytosine

T=thymine

X=T or C if Y is A or G

X=C if Y is C or T

Y=A, G, C or T if X is C

Y=A or G if X is T

W=C or A if Z is A or G

W=C if Z is C or T

Z=A, G, C or T if W is C

Z=A or G if W is A

QR=TC if S is A, G, C or T; alternatively QR=AG if S is T or C

J=A or G

K=T or C

L=A, T, C or G

M=A, C or T

The above shows that the novel amino acid sequence of the M7 toxin, andother useful proteins, can be prepared by equivalent nucleotidesequences encoding the same amino acid sequence of the proteins.Accordingly, the subject invention includes such equivalent nucleotidesequences. In addition it has been shown that proteins of identifiedstructure and function may be constructed by changing the amino acidsequence if such changes do not alter the protein secondary structure(Kaiser, E. T. and Kezdy, F. J. [1984] Science 223:249-255). Thus, thesubject invention includes mutants of the amino acid sequence depictedherein which do not alter the protein secondary structure, or if thestructure is altered, the biological activity is retained to somedegree.

EXAMPLE 3 Production of M-7 Toxin Protein by Clone pCH-B3

A 20 liter culture of pCH-B3 (L-broth with 70 μg/ml Ampicillin) wasgrown in a fermenter and harvested at OD600=3.35. The cell pellet waswashed with water and resuspended in 500 ml glycine buffer (0.1 Mglycine, pH 8.0 with tris base) containing 2 g lysozyme, 1 mM PMSF(phenylmethylsulfonyl fluoride), 1 mM TPCK (1-tosylamide-2-phenylethylchloromethyl ketone), and 500 μg DNase I and incubated at roomtemperature for 30 min. The pH was then raised to 10 with NaOH and thecells were further ruptured in a bead beater (Biospec Products,Bartlesville, OK) on ice with four 30 second bursts 5 min apart. Theextract was then centrifuged at 10,000 x g for 30 min.

EXAMPLE 4 Isolation and Purification of M-7 Toxin Protein Produced byClone pCH-B3

The protein from pCH-B3 was purified using affinity chromatography(Cuatrecasa, P. and Anfinsen, C. B. [1971] Meth. Enzymology Vol. 22 [ed.W. B. Jacoby] Acad. Press, N.Y.) as follows: Sepharose was activatedwith cyanogen bromide as described by Cuatrecasa and Anfinsen. Rabbitanti-M-7 crystal serum was added to the activated Sepharose andincubated overnight at room temperature with constant agitation. Theaffinity resign was then washed with 1% ethanolamine, 3 M NaCl, pH 9.2,and then with TBS (0.02 M tris-HCl, 0.07 M NaCl, pH 7.5) containing0.02% sodium azide. The column was equilibrated in 0.1 M glycine pH 10(with tris base) containing 1 mM EDTA (ethylenediaminetetraacetic acid),1 mM PMSF, 1 mM TPCK, and 0.02% sodium azide. The E. coli extract,prepared above, was loaded onto the column and recirculated for 64 hr at4° C. The extract was washed from the column with 1 M NaCl and 0.1 Mglycine-tris pH 10, and the bound M-7 toxin was removed from the columnwith 3 M sodium perchlorate, 0.1 M glycine-tris pH 10. The M-7 toxin wasthen dialyzed against water and concentrated (MicroPro D: Con, PierceChem. Co., Rockford, Ill.).

The purified M-7 toxin can be administered (applied) to vegetationsusceptible to infestation by beetles of the order Coleoptera to protectthe vegetation. Advantageously, the M-7 toxin will be madeenvironmentally stable by use of suitable coatings well known to personsskilled in the art.

EXAMPLE 5 Subcloning and Expression of M-7 Toxin Gene into Pseudomonasfluorescens

The 5.8 kb DNA fragment carrying the M-7 toxin gene was excised fromplasmid pCH-B3 with BamHI, purified, and subcloned into the BamHI siteof the plasmid pRO1614. Pseudomonas fluorescens was transformed withthis plasmid. The expression of M-7 toxin by recombinant Pseudomonascells was verified by its identification on a western blot.

EXAMPLE 6 Testing of B. thuringiensis strain san diego NRRL B-15939Spores and Crystal

B. thuringiensis strain san diego NRRL B-15939 spores and crystal,obtained as described above, were tested against various insects. Theinsect species tested and a summary of the results are listed in Table1.

The method used to test for D. undecimpunctata (WSCB) activity consistedof spraying a spore/crystal suspension onto leaf discs of lettuce in aspray tower apparatus. (The larvae of this species are reared on lettuceleaves.) The spray was dried in a laminar flow hood and placed in acontainer on moist filter paper. Ten larvae of WSCB were added and thecontainers were incubated at 25° C. and 14 hr photoperiod. Fresh treateddiscs were added as needed. Inhibition of feeding was noted andmortality was recorded at 5 and 7 days. Results of 2 bioassays are givenin Table 2.

In order to test the M-7 toxin for activity against Pyrrhalta luteola(elm leaf beetle), a suspension of solubilized protein from M-7 crystalswas applied to elm leaves. The dried leaves were then placed in acontainer on moist sand. Five to ten larvae of P. luteola were added andthe containers were incubated at room temperature. Mortality wasrecorded at 3 and 5 days. An LC₅₀ of 120 ng toxin/cm² of leaf surfacewas calculated from these assays.

                                      TABLE A                                     __________________________________________________________________________    Nucleotide Sequence Encoding the Bacillus thuringiensis                       strain san diego Toxin Gene                                                   __________________________________________________________________________                                       ATGA ATCCGAACAA                            TCGAAGTGAA                                                                              CATGATACAA                                                                              TAAAAACTAC                                                                              TGAAAATAAT                                                                              GAGGTGCCAA                            CTAACCATGT                                                                              TCAATATCCT                                                                              TTAGCGGAAA                                                                              CTCCAAATCC                                                                              AACACTAGAA                            GATTTAAATT                                                                              ATAAAGAGTT                                                                              TTTAAGAATG                                                                              ACTGCAGATA                                                                              ATAATACGGA                            AGCACTAGAT                                                                              AGCTCTACAA                                                                              CAAAAGATGT                                                                              CATTCAAAAA                                                                              GGCATTTCCG                            TAGTAGGTGA                                                                              TCTCCTAGGC                                                                              GTAGTAGGTT                                                                              TCCCGTTTGG                                                                              TGGAGCGCTT                            GTTTCGTTTT                                                                              ATACAAACTT                                                                              TTTAAATACT                                                                              ATTTGGCCAA                                                                              GTGAAGACCC                            GTGGAAGGCT                                                                              TTTATGGAAC                                                                              AAGTAGAAGC                                                                              ATTGATGGAT                                                                              CAGAAAATAG                            CTGATTATGC                                                                              AAAAAATAAA                                                                              GCTCTTGCAG                                                                              AGTTACAGGG                                                                              CCTTCAAAAT                            AATGTCGAAG                                                                              ATTATGTGAG                                                                              TGCATTGAGT                                                                              TCATGGCAAA                                                                              AAAATCCTGT                            GAGTTCACGA                                                                              AATCCACATA                                                                              GCCAGGGGCG                                                                              GATAAGAGAG                                                                              CTGTTTTCTC                            AAGCAGAAAG                                                                              TCATTTTCGT                                                                              AATTCAATGC                                                                              CTTCGTTTGC                                                                              AATTTCTGGA                            TACGAGGTTC                                                                              TATTTCTAAC                                                                              AACATATGCA                                                                              CAAGCTGCCA                                                                              ACACACATTT                            ATTTTTACTA                                                                              AAAGACGCTC                                                                              AAATTTATGG                                                                              AGAAGAATGG                                                                              GGATACGAAA                            AAGAAGATAT                                                                              TGCTGAATTT                                                                              TATAAAAGAC                                                                              AACTAAAACT                                                                              TACGCAAGAA                            TATACTGACC                                                                              ATTGTGTCAA                                                                              ATGGTATAAT                                                                              GTTGGATTAG                                                                              ATAAATTAAG                            AGGTTCATCT                                                                              TATGAATCTT                                                                              GGGTAAACTT                                                                              TAACCGTTAT                                                                              CGCAGAGAGA                            TGACATTAAC                                                                              AGTATTAGAT                                                                              TTAATTGCAC                                                                              TATTTCCATT                                                                              GTATGATGTT                            CGGCTATACC                                                                              CAAAAGAAGT                                                                              TAAAACCGAA                                                                              TTAACAAGAG                                                                              ACGTTTTAAC                            AGATCCAATT                                                                              GTCGGAGTCA                                                                              ACAACCTTAG                                                                              GGGCTATGGA                                                                              ACAACCTTCT                            CTAATATAGA                                                                              AAATTATATT                                                                              CGAAAACCAC                                                                              ATCTATTTGA                                                                              CTATCTGCAT                            AGAATTCAAT                                                                              TTCACACGCG                                                                              GTTCCAACCA                                                                              GGATATTATG                                                                              GAAATGACTC                            TTTCAATTAT                                                                              TGGTCCGGTA                                                                              ATTATGTTTC                                                                              AACTAGACCA                                                                              AGCATAGGAT                            CAAATGATAT                                                                              AATCACATCT                                                                              CCATTCTATG                                                                              GAAATAAATC                                                                              CAGTGAACCT                            GTACAAAATT                                                                              TAGAATTTAA                                                                              TGGAGAAAAA                                                                              GTCTATAGAG                                                                              CCGTAGCAAA                            TACAAATCTT                                                                              GCGGTCTGGC                                                                              CGTCCGCTGT                                                                              ATATTCAGGT                                                                              GTTACAAAAG                            TGGAATTTAG                                                                              CCAATATAAT                                                                              GATCAAACAG                                                                              ATGAAGCAAG                                                                              TACACAAACG                            TACGACTCAA                                                                              AAAGAAATGT                                                                              TGGCGCGGTC                                                                              AGCTGGGATT                                                                              CTATCGATCA                            ATTGCCTCCA                                                                              GAAACAACAG                                                                              ATGAACCTCT                                                                              AGAAAAGGGA                                                                              TATAGCCATC                            AACTCAATTA                                                                              TGTAATGTGC                                                                              TTTTTAATGC                                                                              AGGGTAGTAG                                                                              AGGAACAATC                            CCAGTGTTAA                                                                              CTTGGACACA                                                                              TAAAAGTGTA                                                                              GACTTTTTTA                                                                              ACATGATTGA                            TTCGAAAAAA                                                                              ATTACACAAC                                                                              TTCCGTTAGT                                                                              AAAGGCATAT                                                                              AAGTTACAAT                            CTGGTGCTTC                                                                              CGTTGTCGCA                                                                              GGTCCTAGGT                                                                              TTACAGGAGG                                                                              AGATATCATT                            CAATGCACAG                                                                              AAAATGGAAG                                                                              TGCGGCAACT                                                                              ATTTACGTTA                                                                              CACCGGATGT                            GTCGTACTCT                                                                              CAAAAATATC                                                                              GAGCTAGAAT                                                                              TCATTATGCT                                                                              TCTACATCTC                            AGATAACATT                                                                              TACACTCAGT                                                                              TTAGACGGGG                                                                              CACCATTTAA                                                                              TCAATACTAT                            TTCGATAAAA                                                                              CGATAAATAA                                                                              AGGAGACACA                                                                              TTAACGTATA                                                                              ATTCATTTAA                            TTTAGCAAGT                                                                              TTCAGCACAC                                                                              CATTCGAATT                                                                              ATCAGGGAAT                                                                              AACTTACAAA                            TAGGCGTCAC                                                                              AGGATTAAGT                                                                              GCTGGAGATA                                                                              AAGTTTATAT                                                                              AGACAAAATT                            GAATTTATTC                                                                              CAGTGAAT                                                            __________________________________________________________________________

                                      TABLE B                                     __________________________________________________________________________    Deduced Amino Acid Sequence of Bacillus thuringiensis                         strain san diego Toxin                                                        __________________________________________________________________________     ##STR1##                                                                      ##STR2##                                                                      ##STR3##                                                                      ##STR4##                                                                      ##STR5##                                                                      ##STR6##                                                                      ##STR7##                                                                      ##STR8##                                                                      ##STR9##                                                                      ##STR10##                                                                     ##STR11##                                                                     ##STR12##                                                                     ##STR13##                                                                     ##STR14##                                                                     ##STR15##                                                                     ##STR16##                                                                     ##STR17##                                                                     ##STR18##                                                                     ##STR19##                                                                     ##STR20##                                                                     ##STR21##                                                                     ##STR22##                                                                     ##STR23##                                                                     ##STR24##                                                                     ##STR25##                                                                     ##STR26##                                                                     ##STR27##                                                                     ##STR28##                                                                     ##STR29##                                                                     ##STR30##                                                                     ##STR31##                                                                     ##STR32##                                                                    Ile Pro Val Asn                                                               __________________________________________________________________________

                                      TABLE 1                                     __________________________________________________________________________    Insects Evaluated for Susceptibility to Bacillus thuringiensis strain san     diego                                                                         Order  Family  Species  Common Name                                                                            Stages Tested                                                                        Activity                              __________________________________________________________________________    Coleoptera                                                                           Chrysomelidae                                                                         Diabrotica                                                                             Western spotted                                                                        Adult, larva                                                                         +                                                    undecimpunctata                                                                        cucumber beetle                                                      Pyrrhalta                                                                              Elm leaf beetle                                                                        Adult, larva                                                                         ++++                                                 luteola                                                                       Haltica  --       Adult, larva                                                                         +++                                                  tombacina                                                             Curculionidae                                                                         Otiorhynchus                                                                           Black vine weevil                                                                      Larva  ++                                                   sulcatus                                                              Tenebrionidae                                                                         Tenebrio Yellow mealworm                                                                        Larva  ++                                                   molitor                                                                       Tribolium                                                                              Red flour beetle                                                                       Adult, larva                                                                         -                                                    castaneum                                                             Dermestidae                                                                           Attagenus                                                                              --       Larva  -                                                    megatoma                                                              Ptinidae                                                                              Gibbium  --       Adult  -                                                    psylloides                                                     Diptera                                                                              Culicidae                                                                             Aedes    Yellow fever                                                                           Larva  -                                                    aegypti  mosquito                                              Lepidoptera                                                                          Noctuidae                                                                             Spodoptera                                                                             Beet armyworm                                                                          Larva  -                                                    exigua                                                                        Trichoplusia                                                                           Cabbage looper                                                                         Larva  -                                                    ni                                                             __________________________________________________________________________

                  TABLE 2                                                         ______________________________________                                        Results of 2 Bioassays of Bacillus thuringiensis M-7 Against Second           Instar Diabrotica undecimpunctata U. at 7 Days Post-Inoculation                              Avg. no.                                                                      leaf discs                                                     Treatment      consumed/rep.                                                                             % Mortality                                        ______________________________________                                        Exp 1 Control  3            7.5 ± 15.0                                     4.3 × 10.sup.7 spores/ml                                                               <1          27.5 ± 9.6                                      4.3 × 10.sup.8 spores/ml                                                               0           62.5 ± 26.3                                     Exp 2 Control  1           12.5 ± 12.6                                     1 × 10.sup.6 spores/ml                                                                 <1          30.0 ± 8.2                                      1 × 10.sup.7 spores/ml                                                                 0           50.0 ± 21.6                                     ______________________________________                                    

We claim:
 1. DNA encoding a toxin, having the nucleotide sequence, asfollows: ##STR33## and equivalent nucleotide sequences coding formolecules with the following amino acid sequence: ##STR34##